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- EMDB-2098: Cryo-electron microscopy reconstruction of doublecortin-stabilise... -

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Basic information

Entry
Database: EMDB / ID: EMD-2098
TitleCryo-electron microscopy reconstruction of doublecortin-stabilised microtubules in presence of kinesin
Map dataReconstruction of doublecortin-stabilised microtubules decorated with kinesin motor domain (rigor)
Sample
  • Sample: Doublecortin-stabilised microtubules decorated with kinesin motor domains
  • Protein or peptide: Tubulin alpha-1D chain
  • Protein or peptide: Doublecortin
  • Protein or peptide: Tubulin beta-2B chain
  • Protein or peptide: Kinesin motor domain
Keywordsdoublecortin / kinesin / microtubule / Microtubule-Associated Protein
Function / homology
Function and homology information


RHO GTPases activate KTN1 / Kinesins / regulation of modification of synapse structure, modulating synaptic transmission / plus-end-directed vesicle transport along microtubule / cytoplasm organization / COPI-dependent Golgi-to-ER retrograde traffic / cytolytic granule membrane / anterograde dendritic transport of neurotransmitter receptor complex / positive regulation of vesicle fusion / mitocytosis ...RHO GTPases activate KTN1 / Kinesins / regulation of modification of synapse structure, modulating synaptic transmission / plus-end-directed vesicle transport along microtubule / cytoplasm organization / COPI-dependent Golgi-to-ER retrograde traffic / cytolytic granule membrane / anterograde dendritic transport of neurotransmitter receptor complex / positive regulation of vesicle fusion / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / MHC class II antigen presentation / positive regulation of intracellular protein transport / ciliary rootlet / lysosome localization / positive regulation of potassium ion transport / JUN kinase binding / plus-end-directed microtubule motor activity / vesicle transport along microtubule / positive regulation of axon guidance / kinesin complex / microtubule motor activity / centrosome localization / mitochondrion transport along microtubule / microtubule lateral binding / stress granule disassembly / natural killer cell mediated cytotoxicity / synaptic vesicle transport / endocytic vesicle / positive regulation of insulin secretion involved in cellular response to glucose stimulus / postsynaptic cytosol / axonal growth cone / microtubule-based process / phagocytic vesicle / axon cytoplasm / dendrite cytoplasm / axon guidance / positive regulation of synaptic transmission, GABAergic / regulation of membrane potential / positive regulation of protein localization to plasma membrane / hippocampus development / cellular response to type II interferon / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / microtubule cytoskeleton / microtubule binding / vesicle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / neuron projection / hydrolase activity / protein heterodimerization activity / GTPase activity / GTP binding / protein-containing complex binding / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / metal ion binding / identical protein binding / cytoplasm
Similarity search - Function
Doublecortin domain / Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin ...Doublecortin domain / Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Tubulin alpha-1D chain / Kinesin-1 heavy chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesBos taurus (domestic cattle) / Homo sapiens (human) / Rattus norvegicus (Norway rat)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 8.2 Å
AuthorsLiu JS / Schubert CR / Fu X / Fourniol FJ / Jaiswal JK / Houdusse A / Stultz CM / Moores CA / Walsh CA
CitationJournal: J Cell Biol / Year: 2010
Title: Template-free 13-protofilament microtubule-MAP assembly visualized at 8 A resolution.
Authors: Franck J Fourniol / Charles V Sindelar / Béatrice Amigues / Daniel K Clare / Geraint Thomas / Mylène Perderiset / Fiona Francis / Anne Houdusse / Carolyn A Moores /
Abstract: Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed ...Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX's unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin-tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX's exquisite binding selectivity uncovers important insights into regulation of cellular MTs.
History
DepositionMay 10, 2012-
Header (metadata) releaseMay 17, 2012-
Map releaseAug 15, 2012-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.55
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.55
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4atx
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4atx
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2098.jpg

Downloads & links

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Map

FileDownload / File: emd_2098.map.gz / Format: CCP4 / Size: 1.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of doublecortin-stabilised microtubules decorated with kinesin motor domain (rigor)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 101 pix.
= 282.8 Å		2.8 Å/pix.
x 50 pix.
= 140. Å		2.8 Å/pix.
x 85 pix.
= 238. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.55 / Movie #1: 1.55
Minimum - Maximum-8.071179389999999 - 11.104262350000001
Average (Standard dev.)0.54360223 (±1.79660368)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin416675
Dimensions5085101
Spacing5085101
CellA: 238.0 Å / B: 140.0 Å / C: 282.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z8550101
origin x/y/z0.0000.0000.000
length x/y/z238.000140.000282.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S123
start NC/NR/NS664175
NC/NR/NS8550101
D min/max/mean-8.07111.1040.544

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Supplemental data

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Sample components

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Entire : Doublecortin-stabilised microtubules decorated with kinesin motor...

EntireName: Doublecortin-stabilised microtubules decorated with kinesin motor domains
Components
  • Sample: Doublecortin-stabilised microtubules decorated with kinesin motor domains
  • Protein or peptide: Tubulin alpha-1D chain
  • Protein or peptide: Doublecortin
  • Protein or peptide: Tubulin beta-2B chain
  • Protein or peptide: Kinesin motor domain

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Supramolecule #1000: Doublecortin-stabilised microtubules decorated with kinesin motor...

SupramoleculeName: Doublecortin-stabilised microtubules decorated with kinesin motor domains
type: sample / ID: 1000 / Oligomeric state: 13-protofilament microtubule / Number unique components: 4

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Macromolecule #1: Tubulin alpha-1D chain

MacromoleculeName: Tubulin alpha-1D chain / type: protein_or_peptide / ID: 1 / Oligomeric state: Heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (domestic cattle) / synonym: Cattle / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightTheoretical: 50 KDa
SequenceInterPro: Alpha tubulin

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Macromolecule #2: Doublecortin

MacromoleculeName: Doublecortin / type: protein_or_peptide / ID: 2 / Name.synonym: DCX / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac
SequenceInterPro: Doublecortin domain

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Macromolecule #3: Tubulin beta-2B chain

MacromoleculeName: Tubulin beta-2B chain / type: protein_or_peptide / ID: 3 / Oligomeric state: Heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (domestic cattle) / synonym: Cattle / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightTheoretical: 50 KDa
SequenceInterPro: Beta tubulin, autoregulation binding site

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Macromolecule #4: Kinesin motor domain

MacromoleculeName: Kinesin motor domain / type: protein_or_peptide / ID: 4 / Details: mutant T93N / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Norway rat
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
Details: 20mM Pipes, 1mM EGTA, 3mM MgCl2, 1mM TCEP, 0.5mM GTP
StainingType: NEGATIVE / Details: cryo-EM
GridDetails: 300 mesh lacey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
DateSep 1, 2009
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Number real images: 63 / Average electron dose: 15 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.76 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticles were aligned using Spider and Frealign (Sindelar and Downing, 2010)
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.2 Å
Applied symmetry - Helical parameters - Δ&Phi: 27.7 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN
Details: Approximately 168,000 asymmetric units were averaged together in the final map
CTF correctionDetails: done with FREALIGN

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Atomic model buiding 1

Initial modelPDB ID:

2xrp


Chain - #0 - Chain ID: E / Chain - #1 - Chain ID: F
SoftwareName: Chimera
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross correlation
Output model

PDB-4atx:
Rigor kinesin motor domain with an ordered neck-linker, docked on tubulin dimer, modelled into the 8A cryo-EM map of doublecortin- microtubules decorated with kinesin

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Atomic model buiding 2

Initial modelPDB ID:

1bg2


Chain - Chain ID: A
SoftwareName: Chimera, Flex-EM
DetailsProtocol: Flexible fitting. Kinesin neck-linker (aa 324-329) was modeled into the EM density, and its position refined using Flex-EM, along with loop 2, loop 9 and helix alpha6
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross correlation
Output model

PDB-4atx:
Rigor kinesin motor domain with an ordered neck-linker, docked on tubulin dimer, modelled into the 8A cryo-EM map of doublecortin- microtubules decorated with kinesin

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